Eukaryotic chromosome segregation requires kinetochores, organelles that assemble on condensing chromosomes to form dynamic attachment sites for spindle microtubules. Errors in kinetochore function contribute to chromosomal instability during tumorigenesis and potentially also to birth defects. Because of their specific roles in cell division, kinetochore components are attractive targets for anti-mitotic chemotherapy. Molecular analysis of kinetochores in metazoans has been limited by their essential nature, rarity of their constituents, and difficulties in translating mechanochemical functions into biochemical assays. Two central unanswered questions are: (1) how is the localized region of the chromosome where the kinetochore assembles specified? and (2) how is the initiation of kinetochore assembly translated into the formation of an interface that interacts with spindle microtubules to direct chromosome segregation? The goal of the proposed work is to address these questions using functional analysis in the C. elegans embryo, and extend some of the studies to human cells. The work will capitalize on the unique access provided by the one-cell stage C. elegans embryo for analyzing the function of essential gene products which, in combination with genomics and biochemistry, is rapidly providing a comprehensive component list for kinetochores. In addition, current assays provide a powerful classification scheme to place newly identified as well as known proteins into specific functional groups and to define their site of action within the substructure of the kinetochore. Kinetochore specification is intimately associated with CENP-A, a centromere-specific histone H3 variant. The first specific aim is directed towards defining the mechanism of CENP-A deposition following fertilization in C. elegans, particularly to distinguish between inheritance versus de novo deposition. The second aim will continue this theme to analyze a protein identified by functional genomics as a likely central player in CENP-A targeting and organization. The third aim will focus on a conserved multi-subunit complex identified by a combination of genomics and biochemistry as playing a key role in propagating initiation of kinetochore assembly to form the interface with spindle microtubules. The final aim will focus on other conserved outer kinetochore proteins to define their specific roles at the interface with spindle microtubules in vivo and to investigate their interactions with microtubules in vitro.